Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum

► We expressed soluble form of human Fas ligand (shFasL) in Escherichia coli and Dictyostelium discoideum, respectively. ► We developed a newly completely synthetic medium for high cell density cultivation of D. discoideum. ► We used Ni-NTA affinity chromatography to purify the recombinant shFasLs expressed by the two different expression systems. ► The two kinds of recombinant shFasLs showed similar biological activities in inducing apoptosis in Fas-expressing cells. Human Fas ligand (hFasL) is a type II membrane protein that induces apoptosis in the Fas-bearing cells. Its special biological activity has the potential for the therapeutic use as an anti-cancer agent directed at enhancing apoptosis in tumor cells. In this study Escherichia coli and eukaryotic Dictyostelium discoideum were used to produce a soluble form of hFasL in large amounts. An expression vector for hFasL production in E. coli was constructed based on plasmid pET32a(+). By cultivation of the hFasL-producing E. coli clone on LB medium and induction with IPTG, a hFasL concentration of 1.0mgL−1 was achieved. D. discoideum strain AX3-hFasL-H was cultured in a conventional stirred bioreactor on an improved synthetic medium using a simple fed-batch strategy, and cell densities of up to 8.3×107cells/mL and a maximum hFasL concentration of 420μg/L were obtained. Using Ni-NTA affinity chromatography purification, two kinds of recombinant hFasLs from E. coli and D. discoideum were purified with a purity of 94% and 90%, respectively. They showed similar biological activities in inducing apoptosis in Fas-expressing cells.